An anti inflammatory role for CEBPδ in human brain pericytes

An anti inflammatory role for C/EBPδ in human brain pericytes 1Scientific RepoRts | 5 12132 | DOi 10 1038/srep12132 www nature com/scientificreports An anti inflammatory role for C/EBPδ in human brain[.]

An anti-inflammatory role for

Justin Rustenhoven 1,4 , Emma L Scotter 1,4 , Deidre Jansson 1,2,4 , Dan T Kho 1,4 , Robyn L Oldfield 5 , Peter S Bergin 4,6 , Edward W Mee 4,6 , Richard L M Faull 3,4 , Maurice A Curtis 3,4 , Scott E Graham 1,4 , Thomas I-H Park 1,4 & Mike Dragunow 1,2,4 Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes Cellular inflammatory responses are orchestrated

by transcription factors but information on transcriptional control in pericytes is lacking Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number

of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion

in human brain pericytes by interleukin-1β (IL-1β) To investigate the function of the induced C/EBPδ

in pericytes we used siRNA to knockdown IL-1β-induced C/EBPδ expression C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry Our results show that knock-down

of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response The induction

of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.

Neuroinflammation contributes to the development and progression of epilepsy1, traumatic brain inju-ries2, stroke3 and many neurodegenerative diseases4 such as motor neuron disease and Alzheimer’s disease Microglia, and to a lesser extent astrocytes, are believed to be the primary initiators of neuroin-flammation and can promote neuronal loss through the secretion of neurotoxic molecules5,6 In addition

to brain glia, pericytes also contribute to the inflammatory response7–10 Brain pericytes are situated surrounding and contacting endothelial cells of brain capillaries and together with astrocytes, neurons and microglia form the neurovascular unit11 Pericyte vascular coverage is essential for the formation and maintenance of the blood-brain barrier (BBB) and the regulation of cerebral blood flow highlighting its importance in central nervous system (CNS) homeostasis12–14

Like brain glia, pericytes can also respond to a range of immunogenic stimuli to induce pro-inflammatory molecules including cytokines e.g., interleukin-6 and 8 (IL-6 and IL-8), chemokines such as monocyte chemoattractant protein-1 (MCP-1) and interferon gamma-induced protein-10 (IP-10) and adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhe-sion molecule-1 (VCAM-1)8–10 Induction of these mediators can promote peripheral immune cell infil-tration15–17, as well as local microglial cell migration18, proliferation19,20 and activation21 enhancing the pro-inflammatory phenotype of the brain and potentially contributing to neuronal loss

Cellular inflammatory responses are orchestrated largely through transcription factor mediated gene expression22 By virtue of conserved promoter/enhancer DNA sequences, a single transcription factor may regulate the expression of numerous inflammatory genes making them an attractive target

1 Department of Pharmacology and Clinical Pharmacology 2 Gravida National Centre for Growth and Development

3 Department of Anatomy with Radiology 4 Centre for Brain Research 5 The University of Auckland, 1023, Auckland, New Zealand 6 Lab Plus, Auckland City Hospital, 1023, Auckland, New Zealand Correspondence and requests for materials should be addressed to M.D (email: m.dragunow@auckland.ac.nz)

Received: 06 November 2014

Accepted: 01 June 2015

Published: 13 July 2015

OPEN

for anti-inflammatory interventions Involvement of the prototypical inflammatory transcription factor nuclear factor-kappa B (NF-kB) has previously been identified in pericyte activation8,10 However, evi-dence regarding further transcription factor involvement in pericyte inflammatory responses is currently lacking and warrants further investigation

A role in mediating pro-inflammatory gene expression has recently been observed with several mem-bers of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, particularly with C/EBPα 23, C/EBPβ 24 and C/EBPδ 25,26 C/EBPs are members of the bZIP family of transcription factors and many inflammatory genes include CCAAT binding motifs in their promoter/enhancer region27 C/EBP family members require dimerisation for DNA binding and do so by forming homodimers or heterodimers with other C/EBP family members, or associated transcription factors including NF-kB28

In response to inflammatory stimuli many tissues demonstrate induction of C/EBPδ expression In the Alzheimer’s brain and in spinal cord of amyotrophic lateral sclerosis (ALS) patients, both of which have a significant inflammatory component, enhanced C/EBPδ protein expression has been observed

in astrocytes and microglia respectively26,29 The functional effects of this induction however remain unclear Studies utilising rodent glia have suggested C/EBPδ has a pro-inflammatory role in the brain through enhancement of inflammatory gene transcription25,26 Indeed this has also been seen in other tissues, including the liver and lung where attenuation of C/EBPδ expression dampened inflammatory responses30–32 However, C/EBPδ induction has also been shown to inhibit pro-inflammatory gene expression in the pancreas33 whilst C/EBPδ deficiency enhanced tubulointerstitial fibrosis, a renal con-dition with an inflammatory component34 Furthermore, induction of a closely related family member C/EBPβ has both anti-inflammatory24 and pro-inflammatory roles in the brain35 As such, it appears C/ EBP family members including C/EBPδ act in a cell and context dependant manner allowing it to dif-ferentially respond to the cell’s situation

Whilst the pericyte contribution to neuroinflammation is being increasingly studied, little is under-stood regarding inflammation-related gene transcription in these cells and to date the role of C/EBPδ has not been studied in human brain pericytes We therefore sought to investigate the function of C/EBPδ in human brain pericyte mediated inflammatory responses

Results

Characterisation of adult human brain pericyte cultures. Immunocytochemical analysis of early passage cell cultures obtained from human middle temporal gyrus tissue reveals a mixed population of astrocytes, microglia and pericytes as described previously10 Under our in vitro conditions microglia

and astrocytes do not proliferate and are diluted out in subsequent passages To ensure no contamina-tion from brain glia in pericyte cultures these were grown until passage five before use Late passage cultures showed positive immunocytochemical staining for the pericyte markers alpha smooth mus-cle actin (α SMA; Fig. 1a), platelet derived growth factor receptor beta (PDGFRβ ; Fig. 1b) and neural/ glial antigen 2(NG2; Fig. 1c) as well as the fibroblast markers prolyl-4-hydroxylase (P4H; Fig. 1d) and fibronectin (Fig. 1e) There were no cells positive for the microglia marker CD45 or the astrocyte marker glial fibrillary acidic protein (GFAP; Fig. 1f) at passage five onwards Positive controls of GFAP (Fig. 1g) and CD45 (Fig. 1h) staining at passage two are shown

C/EBPδ is induced in human brain pericytes by IL-1β/IFNγ Microarray analyis of human brain pericytes treated with IL-1β /IFNγ has previously revealed an induction of numerous inflammatory genes10 Due to the ability of the C/EBP family of transcription factors to modify cellular inflammatory responses, we chose to investigate the induction of three members C/EBPα , C/EBPβ and C/EBPδ in this dataset By microarray analysis, C/EBPδ was found to be significantly increased by an IL-1β /IFNγ treatment (p < 0.001) whilst C/EBPα (p > 0.05) and C/EBPβ (p < 0.05) were not affected (Fig. 2a) Real time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on inde-pendent RNA samples, which confirmed the induction of C/EBPδ (5.33 ± 0.69 fold; p < 0.001), whereas

no significant change in C/EBPα (0.74 ± 0.18 fold; p > 0.05) or C/EBPβ (1.18 ± 0.15 fold; p > 0.05) was observed (Fig. 2b), consistent with the microarray data

C/EBPδ is differentially induced by IL-1β, IFNγ and LPS Having observed an induction of C/ EBPδ with a combination of IL-1β and IFNγ we sought to investigate how individual inflammatory stimuli affect this response IL-1β , IFNγ and LPS were investigated based on prior evidence for their involvement in pericyte inflammatory responses As determined by immunocytochemistry the basal expression of C/EBPδ in pericyte cultures is low (9.85 ± 1.07%; Fig.  3a,b) Enhanced nuclear expres-sion was observed with IL-1β alone (49.85 ± 3.36%; p < 0.001) and a combination of IL-1β and IFNγ (54.15 ± 2.96%; p < 0.001) Neither IFNγ (18.14 ± 1.80%; p > 0.05) or LPS (18.46 ± 2.57%; p > 0.05) were sufficient to significantly induce C/EBPδ expression (Fig. 3a,b) Western blot analysis revealed a similar trend with both IL-1β alone (7.63 ± 2.52 fold; p < 0.05) and in combination with IFNγ (10.82 ± 1.50 fold; p < 0.01) significantly enhancing C/EBPδ expression whilst IFNγ (3.67 ± 0.77 fold; p > 0.05) and LPS (3.32 ± 0.86 fold; p > 0.05) did not (Fig. 3c,d)

Time course and concentration-dependant induction of C/EBPδ expression As IL-1β treat-ment resulted in the greatest induction of C/EBPδ all subsequent experitreat-ments were performed using

this inflammatory cytokine To further understand the profile of C/EBPδ expression in human brain pericytes, a concentration-response curve of IL-1β induced expression was performed Using immu-nocytochemistry, C/EBPδ was found to be induced by IL-1β in a concentration-dependant manner (Fig. 4a,b) A significant induction was observed by concentrations as low as 0.1 ng/mL IL-1β (vehicle 13.07 ± 1.20%, IL-1β 26.18 ± 1.16%; p < 0.001) with maximal induction by 10 ng/mL (53.73 ± 2.49%;

p < 0.001) Western blotting revealed a significant induction of C/EBPδ using 1 ng/mL (10.12 ± 3.06 fold; p < 0.05) and 10 ng/mL (8.57 ± 0.99 fold; p < 0.05) IL-1β (Fig.  4c,d) In order to investigate the temporal profile of C/EBPδ induction a time-course was performed with 10 ng/mL IL-1β By immuno-cytochemistry C/EBPδ was induced as early as two hours after IL-1β stimulation (vehicle 13.95 ± 0.85%, IL-1β 23.03 ± 1.71%; p < 0.05), maximally induced four hours after treatment (72.83 ± 3.11%; p < 0.001) and remained elevated 48 hours later (34.43% ± 1.45%; p < 0.001; Fig.  4e) Western blotting analysis revealed a similar trend with significant induction of C/EBPδ at two (23.42 ± 5.67 fold; p < 0.05), four

Figure 1 Characterisation of adult human brain pericyte cultures Primary human brain cell cultures

at passage five were stained for Hoechst (blue) and cell specific markers α SMA (a), PDGFRβ (b), NG2 (c), P4H (d), Fibronectin (e), CD45 and GFAP (f) Positive controls of astrocytes (GFAP; g) and microglia (CD45; h) at passage two are included Scale bar = 50 μ m.

Figure 2 C/EBPδ is induced in human brain pericytes by IL-1β/IFNγ Human brain pericytes were

treated with vehicle or 10 ng/mL IL-1β + IFNγ for 24 hours and RNA was extracted Expression of C/ EBPα , C/EBPβ and C/EBPδ was determined by microarray10 (a) and qRT-PCR (b) Data is displayed as average fold change of five independent cases (a) or mean ± SEM of a separate three independent cases (b)

*** = p < 0.001

(27.32 ± 10.55 fold; p < 0.05) and 24 hour treatments (12.45 ± 3.24 fold; p < 0.05) with IL-1β However,

it was not significantly elevated at 48 hours (9.43 + 1.85 fold; p > 0.05; Fig. 4f,g)

Figure 3 C/EBPδ is differentially induced by IFNγ, IL-1β and LPS Human brain pericytes were treated

with vehicle or 10 ng/mL IL-1β , IFNγ , or LPS for 24 hours Representative immunocytochemistry images of

C/EBPδ with treatments are shown (a) The percentage of cells positive for nuclear C/EBPδ was determined

by immunocytochemistry (b) and the intensity of C/EBPδ expression was analysed by western blotting (c,d) Blots are cropped to improve clarity Full-length blots are presented in Supplementary Figure S2 Data

is displayed as mean ± SEM of three independent experiments * = p < 0.05 compared to vehicle control,

** = p < 0.01 compared to vehicle control *** = p < 0.001 compared to vehicle control Scale bar = 100 μ m

Figure 4 Time-course and concentration-dependant induction of C/EBPδ expression Human

brain pericytes were treated with vehicle or 0.01–10 ng/mL IL-1β for 24 hours Representative

immunocytochemistry images of C/EBPδ are shown (a) The percentage of cells positive for nuclear C/ EBPδ was determined by immunocytochemistry (b) and C/EBPδ intensity was analysed by western blotting (c,d) Blots are cropped to improve clarity Full-length blots are presented in Supplementary Figure S2

Human brain pericytes were treated with 10 ng/mL IL-1β for 0–48 hours The percentage of C/EBPδ positive

cells was determined by immunocytochemistry (e) and C/EBPδ intensity was analysed by western blotting (f,g) Data is displayed as mean ± SEM from three independent experiments * = p < 0.05 compared to vehicle control, ** = p < 0.01 compared to vehicle control, *** = p < 0.001 compared to vehicle control Scale bar = 100 μ m

Knockdown of C/EBPδ using siRNA C/EBPδ has been widely reported to modify inflammatory gene expression in various cell types To investigate its effects on human brain pericyte inflammatory responses, a C/EBPδ siRNA construct was employed Transfection of 50 nM C/EBPδ siRNA signifi-cantly reduced IL-1β induced C/EBPδ expression at four hours (control siRNA 62.70 ± 1.18%, C/EBPδ siRNA 18.17 ± 3.07%; p < 0.001), 24 hours (control siRNA 49.63 ± 1.51%, C/EBPδ siRNA 16.14 ± 3.05%;

p < 0.001) and 48 hours (control siRNA 29.26 ± 1.01%, C/EBPδ siRNA 9.71 ± 1.89%; p < 0.001), how-ever, had no effect on basal levels (control siRNA 3.89 ± 0.98%, C/EBPδ siRNA 4.99 ± 0.99%; p > 0.05) as determined by immunocytochemistry (Fig. 5a,b) Using qRT-PCR a reduction in both basal (0.22 ± 0.02 fold; p < 0.001) and IL-1β induced gene expression (control siRNA 4.86 ± 0.62 fold, C/EBPδ siRNA 1.18 ± 0.17; p < 0.001; Fig.  5c) was observed with C/EBPδ siRNA Western blotting analysis revealed

no basal change in C/EBPδ with siRNA treatment (0.05 ± 0.03 fold; p > 0.05), however a significant attenuation of IL-1β induced expression was observed (control siRNA 8.80 ± 1.85 fold, C/EBPδ siRNA 0.37 ± 0.23; p < 0.001; Fig. 5d,e)

C/EBPδ knockdown modifies IL-1β induced inflammatory gene expression Having observed

a reduction in IL-1β induced C/EBPδ expression with siRNA transfection, we examined the effect on a range of inflammatory mediators which have previously been shown to be induced by brain pericytes with inflammatory stimuli C/EBPδ knockdown resulted in increased IL-1β -induced expression of IL-1β (control siRNA 29.74 ± 4.13 fold, C/EBPδ siRNA 55.88 ± 4.99 fold; p < 0.001; Fig.  6a), ICAM-1 (con-trol siRNA 125.20 ± 5.40 fold, C/EBPδ siRNA 295.72 ± 41.51 fold; p < 0.001; Fig. 6b), MCP-1 (con(con-trol siRNA 19.04 ± 1.39 fold, C/EBPδ siRNA 32.59 ± 1.61 fold; p < 0.001; Fig. 6c) and IL-8 (control siRNA 634.81 ± 38.01 fold, C/EBPδ siRNA 1021.12 ± 121.10 fold; p< 0.001; Fig.  6d) In contrast, attenuated expression was observed for SOD2 (control siRNA 56.72 ± 4.47 fold, C/EBPδ siRNA 41.50 ± 1.98 fold;

p < 0.01; Fig. 6e) and COX-2 (control siRNA 6.35 ± 4.86 fold, C/EBPδ siRNA 4.50 ± 1.14 fold; p < 0.001; Fig.  6f), whilst a non-significant decrease was observed for IL-6 (control siRNA 530.91 ± 174.61 fold, C/EBPδ siRNA 369.92 ± 133.60 fold; p > 0.05; Fig. 6g) The basal expression of all inflammatory genes showed no change with C/EBPδ or control siRNA

C/EBPδ knockdown enhances IL-1β induced ICAM-1 and MCP-1 expression In order to determine whether changes at the RNA level correlated with altered protein expression, immunocytochemistry for ICAM-1 and MCP-1 was performed Unstimulated pericytes showed low basal expression of ICAM-1 and this was unaffected by C/EBPδ siRNA (1.00 ± 0.10 AU and 1.82 ± 0.30 AU respectively; Fig. 7a,b,e) A significant induction of ICAM-1 expression was observed with IL-1β treatment at four (104.61 ± 7.98AU;

p < 0.001; Fig. 7e), 24 (518.92 ± 53.80AU; p < 0.001; Fig. 7c,e) and 48 hours (245.31 ± 15.61AU; p < 0.001; Fig. 7e) Compared to the control siRNA, C/EBPδ siRNA further increased IL-1β stimulated ICAM-1 expression at four (230.93 ± 14.85AU; p < 0.01; Fig. 7e), 24 (843.56 ± 128.70AU; p < 0.001; Fig. 7c, e) and

48 hours (394.52 ± 27.55AU; p < 0.01; Fig. 7e) Immunocytochemical analysis of MCP-1 demonstrated low basal levels which were also unaffected by C/EBPδ siRNA (12.77 ± 0.89% and 13.77 ± 1.03% respec-tively; Fig.  7f,g,j) The percentage of MCP-1 positive cells was significantly increased following IL-1β treatment at two (42.73 ± 0.81%; p < 0.001; Fig 7j), four (63.47 ± 1.62%; Fig 7j), 24 (52.51 ± 1.61%; Fig 7h,j) and 48 hours(49.56 ± 2.97%; Fig 7j) Compared to control siRNA, C/EBPδ siRNA further increased IL-1β stimulated MCP-1 expression at each of the measured time point (2 hours, 59.83 ± 1.13%; four hours 80.67 ± 0.83%; 24 hours 73.51 ± 2.25%; 48 hours 70.39 ± 4.15%; p < 0.001; Fig. 7i,j)

C/EBPδ knockdown modifies pericyte secretion of inflammatory mediators Secreted cytokines and chemokines are essential in modulating cellular cross-talk and inflammatory responses

In order to determine whether the observed changed in mRNA and protein expression correlated with increased secretion, cytokine concentrations in the pericytes conditioned media were measured using a cytometric bead array (CBA) Unstimulated pericytes demonstrated basal expression of soluble ICAM-1 (sICAM-1; 3.13 ± 0.75 pg/mL), MCP-1 (4,560.79 ± 143.38 pg/mL) , IL-8 (2,483.51 ± 202.58 pg/mL) and IL-6 (22.75 ± 2.80 pg/mL), which were not significantly altered by C/EBPδ siRNA (ICAM-1 4.15 ± 0.3 pg/ mL; p > 0.05, MCP-1 4,837.74 ± 188.27 pg/mL ; p > 0.05, IL-8 2,345.53 ± 206.12 pg/mL ; p > 0.05 and IL-6 21.25 ± 2.66 pg/mL; p > 0.05; Fig. 8a–d) IL-1β treatment significantly increased the concentration of all measured cytokines in the media (ICAM-1 139.90 ± 11.85 pg/mL; p < 0.001, MCP-1 11,755.35 ± 69.58 pg/ mL; p < 0.001, IL-8 37,616.57 ± 10,815.63 pg/mL; p < 0.001 and IL-6 6,040.13 ± 885.67 pg/mL; p < 0.001; Fig. 8a–d) Compared to the control siRNA condition, C/EBPδ siRNA significantly enhanced the IL-1β induced secretion of ICAM-1 (246.5 ± 10.93 pg/mL; p < 0.001; Fig. 8a) and MCP-1 (14,551.65 ± 205.73 pg/ mL; p < 0.001; Fig. 8b); however, did not alter IL-8 (50,833.75 ± 2,719.64 pg/mL; p > 0.05; Fig. 8c) or IL-6 secretion (7,724.97 ± 705.35 pg/mL; p > 0.05; Fig. 8d)

Discussion

C/EBPδ was found to be induced in human brain pericyte cultures by the pro-inflammatory cytokine IL-1β Several studies have reported induction of C/EBPδ expression in microglia and astrocytes follow-ing inflammation25,26,29, however, C/EBPδ expression in pericytes has not been previously shown Using C/EBPδ specific siRNA we were able to attenuate the induction of pericyte C/EBPδ following immune challenge in order to identify its contribution to the inflammatory response

Figure 5 Knockdown of C/EBPδ using siRNA Human brain pericytes were transfected with 50 nM of

control or C/EBPδ siRNA for 48 hours Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 4–48 hours Representative immunocytochemistry images of C/EBPδ with treatments are shown

(a) and the percentage of cells positive for nuclear C/EBPδ was quantified (b) RNA was extracted following

a six hour treatment with IL-1β and C/EBPδ transcript expression was determined via RT-qPCR (c) Protein

was extracted following a 24 hour treatment with IL-1β and C/EBPδ expression determined by western

blotting (d,e) Blots are cropped to improve clarity Full-length blots are presented in Supplementary Figure

S2 *** = p < 0.001 Scale bar = 100 μ m

Consistent with literature from rodent microglia and astrocytes, C/EBPδ knock-down attenuated COX-2 expression; however it did not significantly alter IL-6 expression25,26 COX-2 is an inducible enzyme that catalyses prostaglandin formation, many of which have roles in inflammation Whilst typ-ically thought to have a pro-inflammatory response, anti-inflammatory roles of prostaglandins are now being acknowledged36 Prostaglandin I2 (PGI2) is the most prominent prostaglandin synthesised by cells

of the vasculature including endothelial cells and vascular smooth muscle cells and is predominantly synthesised via COX-237,38 Furthermore, PGI2 is the major prostaglandin product from retinal pericyte cells39 PGI2 inhibits leukocyte adhesion to vascular endothelium and therefore may attenuate immune infiltration40 In order to understand the specific inflammatory role of COX-2 induction in pericytes the composition of prostaglandins needs to be studied in context of the system Similarly, a dual role in both anti-inflammatory and pro-inflammatory responses has been observed for IL-6 with evidence support-ing a neuroprotective role in the brain41,42 Care should be taken when classifying these as either pro or anti-inflammatory due to their context dependant nature

Attenuation of IL-1β induced SOD-2 expression was also observed with C/EBPδ knock-down Induction of SOD-2 by a range of inflammatory factors has previously been shown, whilst its pro-moter region contains a C/EBP binding site43,44 SOD-2 regulates oxidative stress in cells by converting superoxide into hydrogen peroxide Superoxide ions enhance recruitment of immune cells, precipitate DNA damage and were found to augment behavioural deficits in a mouse model of Alzheimer’s dis-ease45–47 Furthermore, oxidative stress is believed to be a major contributor to neuronal damage in inflammatory conditions and elimination of superoxide formation by SOD-2 is often considered to be anti-inflammatory48 The finding that C/EBPδ enhances SOD-2 induction may indicate an anti-oxidant/ anti-inflammatory role for this transcription factor following inflammation

Figure 6 C/EBPδ knockdown modifies IL-1β induced inflammatory gene expression Human brain

pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for six hours and RNA was extracted The effect of C/EBPδ

knockdown on IL-1β (a), ICAM-1 (b), MCP-1 (c), IL-8(d), SOD2 (e), COX-2 (f) and IL-6 (g) was assayed

by qRT-PCR Data is displayed as mean ± SEM from four independent cases ** = p < 0.01, *** = p < 0.001

Interestingly, C/EBPδ knock-down was also found to enhance the expression of several IL-1β induced inflammatory genes including IL-8, ICAM-1, MCP-1 and IL-1β Two of which, MCP-1 and ICAM-1, were confirmed to be upregulated by immunocytochemical staining, as well as secretions into culture media MCP-1 is a major chemokine involved in the recruitment of monocytes In the context of CNS immune responses, secretions of MCP-1 from brain pericytes could be involved in the trafficking of monocytes from blood into the brain.49,50 Several parenchymal brain cells produce MCP-1 under inflammatory conditions, including microglia, astrocytes and pericytes10 Peripheral immune cell recruitment to the brain may further enhance central inflammatory responses through potentiated inflammatory cytokine production Furthermore, MCP-1 increases both local microglial proliferation, as well as migration to injured sites19 As the brain’s predominant immune cell, increased microglial presence and activation may further worsen inflammatory responses, contributing to neuronal death Indeed, enhanced MCP-1 expression has been observed in several neurological disorders and may worsen disease progression51–53 Following inflammatory responses, the finding that C/EBPδ induction acts to dampen MCP-1 expression

in brain pericytes suggests potential for reducing peripheral immune cell infiltration Interestingly, the only study currently proposing an anti-inflammatory role of C/EBPδ utilised pancreatic beta cells to show that C/EBPδ knock-down enhanced IL-β /IFNγ induced chemokine expression, corroborating the changes we see in MCP-1 expression33 Investigations into the effect of C/EBPδ on brain glia chemokine expression are currently lacking

ICAM-1 is a transmembrane protein involved in stabilising cell-cell interactions15 It is a ligand for the CD11 family of leukocyte adhesion molecules, which are widely expressed and inducible on leukocytes and this interaction aids their attachment to brain vasculature and subsequent migration into tissue54 Under basal conditions ICAM-1 expression in unstimulated pericytes is low; however it was found to be significantly elevated by the pro-inflammatory cytokine IL-1β Expression of ICAM-1 on pericytes has

Figure 7 C/EBPδ knockdown enhances IL-1β induced MCP-1 and ICAM-1 protein expression

Human brain pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours Following transfection, cells were treated with vehicle or 10 ng/mL IL-1β for 2–24 hours and cells fixed and immunostained for ICAM-1 and MCP-1 Representative images of ICAM-1 and MCP-1 immunostaining

respectively with vehicle and control siRNA (a,f), vehicle with C/EBPδ siRNA (b,g), 24 hours IL-1β with control siRNA (c,h) and 24 hours IL-1β with C/EBPδ siRNA are shown (d,i) Intensity of ICAM-1 staining (e) and the percentage of MCP-1 positive cells (j) were determined Data is displayed as mean ± SEM from

three independent experiments ** = p < 0.01, *** = p < 0.001 Scale bar = 100 μ m

previously been shown to aid peripheral immune cell infiltration across tissue vasculature, potentially worsening CNS inflammatory responses8,16,54 The finding that C/EBPδ knockdown enhances ICAM-1 expression on brain pericytes suggests that C/EBPδ induction during immune challenge may be bene-ficial in preventing ICAM-1mediated immune cell infiltration, highlighting another anti-inflammatory role for this transcription factor Interestingly, C/EBPβ knockdown in human astrocytes was also found

to enhance IL-1β induced ICAM-1 expression suggesting that this protein is largely regulated by C/EBP family members24

C/EBPδ knockdown also enhanced IL-1β induced IL-8 and IL-1β gene expression IL-8 is a potent neutrophil chemokine that has previously been observed in brain pericytes and involved in enhanced CNS immune cell infiltration8,55 Like MCP-1 and ICAM-1, it represents another mechanism whereby pericytes can enhance central infiltration of peripheral immune cells Interestingly, the change in IL-8 mRNA expression with C/EBPδ knockdown did not translate to a significant change in protein secre-tion The reasons for this are currently unclear IL-8 was the most concentrated of all secreted proteins measured in culture media and perhaps saturation of secretory pathways was observed at the measured time-point C/EBPδ regulation of IL-8 therefore warrants further investigation

Figure 8 C/EBPδ knockdown modifies pericyte secretion of inflammatory mediators Human brain

pericytes were transfected with 50 nM of control or C/EBPδ siRNA for 48 hours Following transfection cells were treated with vehicle or 10 ng/mL IL-1β for 24 hours and conditioned media collected Concentration of

sICAM-1 (a), MCP-1 (b), IL-8 (c) and IL-6 (d) in media was determined using a multiplex cytometric bead

array Data represent mean ± SEM (n = 4) *** = p < 0.001